This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Under physiological conditions, asparagine (Asn) residues can deamidate spontaneously, generating a mixture of aspartic (Asp) and isoaspartic acid (isoAsp) residues via a succinimide intermediate. In addition, isoAsp residue may also be formed through Asp isomerization, although this occurs at a much slower rate. Differentiation of Asp and isoAsp residues is important as the latter often causes more significant changes in protein conformation and functions, which has been linked to many protein misfolding diseases and other pathological processes. An electron capture dissociation (ECD)-MS/MS based method was established in this laboratory, where diagnostic c+57/z+-57 ions from isoAsp residues were used for the differentiation and relative quantification of the two isomeric forms. This method has been applied to study the Asn deamidation and Asp isomerization in several model systems as summarized below. Asn deamidation and Asp isomerization in A[unreadable] Accumulaion of isoAsp residues in amyloid beta (A[unreadable]) peptides either as a result of Asn deamidation or Asp isomerization has been linked to the pathology of Alzheimer's disease. Several synthetic A[unreadable] fragment peptides, including the most toxic form, A[unreadable] 1-42, were analyzed by ECD. Extensive inter-residue cleavages were obtained, and the diagnostic c+57 and/or z+-57 ions were observed for all isoAsp containing peptides. The applicability of electron ionization dissociation (EID) to isoAsp analysis was also validated by the presence of the isoAsp diagnostic ion in the EID spectrum of an Asp-isomerized A[unreadable] fragment peptide. These results have been published in a recent Analytical Chemistry paper (Sargaeva et al. 2009). Top-down ECD analysis and MS3 study of [unreadable]2-microglobulin protein deamidation Although differentiation of Asp and isoAsp by ECD at the peptide level has been well established, there have been no reports on extending this method to identify isoAsp residue at the protein level. A top-down ECD analysis has several advantages over the bottom-up approach, by eliminating the additional sample processing steps associated with the enzymatic digestion that may introduce artificial deamidations (Li et al. 2008) or cause sample losses. The protein chosen here for the development of a top-down approach for isoAsp analysis is [unreadable]2-microglobulin (B2M), a 12 kDa protein that has significant implication in dialysis-related amyloidosis. The aged B2M (after reductive alkylation) showed ~1 Da mass shift in its mass spectrum, suggesting that one of the Asn residues was deamidated. ECD of the aged B2M produced the isoAsp diagnostic ion, c16+57, unambiguously identifying the Asn17 as the deamidation site. Asn17 is part of a fast deamidating -NG- sequence located on the surface of the protein, making it a particularly facile deamidating site. This experiment demonstrated, for the first time, that the ECD method can be applied directly to the detection of isoAsp at the intact protein level. The top-down approach has its own limitations. An increase in protein size often leads to a decrease in the percentage of inter-residue cleavages, as well as the likelihood of the C[unreadable]-C[unreadable] cleavage necessary for the isoAsp diagnostic ion formation. In addition, as more fragmentation channels become available for larger proteins, ion abundance in any given channel tends to decrease. Finally, the extensive noncovalent interactions present in larger proteins may also prevent fragment ion separation after ECD, further contributing to a decrease in the diagnostic ion abundance. One possible solution to these challenges is to perform ECD analysis on a smaller piece of the protein, generated by a traditional fragmentation method, such as collisionally activated dissociation (CAD). This MS3 approach retains the top-down advantage since it, too, does not require prior enzymatic digestion, while the smaller size of the ECD precursor ion increases the odds for the observation of the isoAsp diagnostic ions. As a proof of principle, ECD was performed on the b22(4+) ion of B2M generated by CAD, and the diagnostic c16+57 ion was observed, with a better S/N ratio than that obtained in ECD of the intact protein. These results will be presented as a poster at the 58th annual ASMS conference (Li et al. 2010). We are currently extending this work to beta-amino acids and to glutamine deamidation studies. Research progresses made in this area are reported below. Characterization of beta-peptides using ECD Beta-peptides are peptides containing [unreadable]-amino acid residues, with their amino groups bonded to the [unreadable]- rather than the [unreadable]-carbon. Beta-peptides may exist in two different forms: a [unreadable]2 linked peptide has its side chain connected to the [unreadable] carbon, and a [unreadable]3 linked peptide has its side chain connected to the [unreadable] carbon. Because of their rare occurrence in nature and resistance to proteolytic degradations, they are being explored as a way to evade antibiotic resistance. Since ECD has been implemented successfully to identify isoAsp residues, which is a [unreadable]3 linked peptide, it may also be applicable to differentiate the [unreadable]2 and [unreadable]3 linked peptides. ECD and electron transfer dissociation (ETD) analyses of several synthetic beta-peptides were performed on a 12 T Bruker solariX FT-ICR mass spectrometer and a Bruker AmaZon ion trap instrument, respectively. The heated glass capillary incorporated in the ionization source of these instruments has made possible generation of doubly charged Q06 peptide (V[unreadable]2A[unreadable]2L[unreadable]2V[unreadable]3A[unreadable]3L[unreadable]3) ions for ExD analysis, which was previously difficult to obtain. Consistent with previous observations in ECD of a substance P analogue, N-C[unreadable] and C[unreadable]-C[unreadable] cleavages at the [unreadable]-amino acid sites were generally absent with the sole exception of isoAsp, highlighting the importance of the adjacent carboxyl group in isoAsp for radical stabilization. In place of the c/z ions, a/y ion formation was greatly enhanced at the N-terminal side of the [unreadable]-amino acid residues, particularly at cyclized [unreadable]-amino acid residues as present in an HIV envelop protein analogue. This could be explained by an alternative ECD mechanism, which is initiated by an electron capture at a protonated amide nitrogen. Subsequent homolytic cleavage can occur either at its N-terminal side to produce a/y ions, or at its C-terminal side to produce c/z ions, the latter of which is inhibited in [unreadable]-peptides because of the instability of the resulting z+ ions. This mechanism is also consistent with the observation of higher a/y ion abundances in ECD of peptides of higher charge states, where amide nitrogen protonation is more likely to occur. It appears that ECD can be used to identify the presence of [unreadable]-amino acids, based on the enhanced a/y cleavage and the diminished c/z cleavage. However, differentiation of the two types of [unreadable]-cleavages was not achieved in the current study. These results will be presented as a poster at the 58th annual ASMS conference (Sargaeva et al. 2010). Differentiation of Glutamic and [unreadable]-glutamic acid residues Although Asn deamidation is the most commonly observed PTM in proteins, glutamine (Gln) may also deamidate under physiological conditions to generate a mixture of glutamic acid (Glu) and [unreadable]-glutamic acid ([unreadable]-Glu) acid. Gln deamidates at a much slower rate, about two orders of magnitude slower compared with its Asn counterpart. Gln deamidation is usually observed in proteins with long turn-over time, such as in eye lens crystallins. Crystallins are highly soluble structure proteins and comprise 90% of lens proteins, which undergo little turnover during their life spans, allowing accumulation of many kinds of modifications. Among these, deamidation is one of the most prevalent, which decreases crystallin solubility, alters lens transparency, and is also involved in cataract formation, a leading cause of blindness. Extensive Gln deamidation has been observed in crystallin proteins. In this study, we explored the possibility of extending the ECD method to differentiate Glu and [unreadable]-Glu, based on the knowledge obtained in isoAsp studies. Continuing from last year's ECD study on a set of synthetic crystallin peptide fragments containing either Glu or [unreadable]-Glu residues, a set of substance P variants were analyzed by ECD to investigate the possible presence of N-terminal diagnostic ions. Although two sets o C-terminal fragments, corresponding to z+-59 and z+-72 ion, exist site specifically at the [unreadable]-Glu residues, only the latter can be used to identify the presence and locate the position of the [unreadable]-Glu residues, because of the presence of z+-59 ions in Glu-containing peptides, albeit without the site-specificity. All N-terminal diagnostic ions, c+57, c+59 and c+72 ions, observed so far, were specific to the Pro-isoGlu sequence. Unlike its Asp counterpart, no diagnostic side-chain loss ions were found in Glu-containing peptides. The presence of Glu residue(s) may be inferred from the observation of a series of zn+-59 ions, although it was neither site specific, nor without interference from the [unreadable]-Glu residues. A manuscript describing these results has been accepted for publication in Analytical Chemistry (Li et al. 2010).